S252: MODULATING HEMATOPOIESIS: ENGINEERING TPO VARIANTS WITH DISTINCT HEMATOPOIETIC FUNCTION
Authors: Zahra Masoumi, Lucie Moss, Julie Tucker, Sophie James, Lora Picton, Naotaka Tsutsumi, William Grey, Chris Garcia, Ian Hitchcock
Journal: HemaSphere (2023)

Background: Thrombopoietin (TPO) is a class I cytokine essential for hematopoietic stem cell (HSC) maintenance and the primary driver of megakaryocyte (MK) differentiation and platelet (PLT) production. Clinically, TPO agonists have emerged as key therapies for the treatment of chronic immune thrombocytopenia and severe aplastic anemia. However, induction of PLT production comes at the cost of increased HSC proliferation, risk of bone marrow fibrosis and a higher incidence of thrombosis, highlighting a critical need to decouple these functions. Here, we used cytokine engineering to generate TPO variants with distinct functional impact in regulating HSC proliferation versus PLT production. Aims: To generate modified TPO variants that alter the downstream signaling and fine-tune the functional hematopoietic outputs. Methods: Site-direct mutagenesis was used to introduce amino acid substitutions in TPO at the low affinity ligand-receptor interface (site 2). Receptor binding affinity was determined by surface plasmon resonance (SPR) and receptor activation assays comparing modified and WT TPO. Initial screening of functional differences was identified by growth and phospho-flow cytometry assays in UT7-TPO cells to select the most promising modifications, which were then generated recombinantly. Further functional assays focused on determining differences in human HSC expansion in vitro and murine HSC proliferation and megakaryocyte differentiation in vivo. Results: Site 2 TPO modifications (TPOmod) were generated and tested by growth assays and phospho-flow cytometry analysis. Among the TPOmod variants, two modifications in K35 (K35A and K35Q) and one in R119 (R119E) demonstrated partial agonist characteristics, supporting cell growth and activating signaling, with maximal stimulation significantly lower than WT TPO. Although binding affinity of TPOmod to TPO receptor was unaltered, levels of active JAK2 and STAT3/5 were dramatically reduced following stimulation by TPOmod, with no significant differences in the activation of AKT, ERK1/2 or CREB, suggesting that it is possible to fine-tune receptor activation through TPO modifications and to direct targeted functional outcomes. To determine functional differences in primary cells, we compared WT TPO with TPOmod in 7-day human HSC expansion cultures. Although WT TPO was able to drive greater overall cell proliferation, TPOmod treatment led to a 2-fold increase in the proportion of EPCR+ HSCs compared to WT TPO. In mice, both WT and TPOmod treatment increased the circulating PLT count to comparable levels in vivo. Critically, only WT TPO stimulated hyperproliferation of MK, MK progenitors and EPCR+ HSCs, whereas TPOmod treatment induced a significant but substantially lower HSC expansion with no changes in MK populations in mice. Summary/Conclusion: By modifying how TPO interacts with TPO receptor, we are able to fine-tune its downstream signalling and, consequently, its functional outcomes. Our data shows that by using a modified ligand, it is possible to uncouple the two primary roles of TPO, HSC expansion and PLT production, highlighting the potential for generating new modified ligands that are more effective at either HSC expansion or platelet production. Keywords: Signaling, Hematopoietic stem cell, Platelet count, Thrombopoietin (TPO)